The invention relates to chemotherapy and DNA repair.
Cancer chemotherapy involves the administration of one or more cytotoxic or cytostatic drugs to a patient. The goal of chemotherapy is to eradicate a substantially clonal population (tumor) of transformed cells from the body of the individual, or to suppress or to attenuate growth of the tumor. Tumors may occur in solid or liquid form, the latter comprising a cell suspension in blood or other body fluid. A secondary goal of chemotherapy is stabilization (clinical management) of the afflicted individual""s health status. Although the tumor may initially respond to chemotherapy, in many instances the initial chemotherapeutic treatment regimen becomes less effective or ceases to impede tumor growth. The selection pressure induced by chemotherapy promotes the development of phenotypic changes that allow tumor cells to resist the cytotoxic effects of a chemotherapeutic drug.
Several chemotherapeutic drugs function by preferentially damaging DNA in actively dividing cells. The treated cells stop proliferating because the damaged genomic DNA is unable to support further mitosis. Types of DNA-damaging chemotherapeutic drugs include those that covalently modify bases (e.g., alkylating agents such as cyclophosphamide) and base analogs (e.g., 5-bromouracil). After chronic exposure to these drugs, tumor cells can become resistant to their effects.
One mechanism by which cells resist chemotherapeutic drugs is modulation of DNA repair processes. For an overview, see, Barrett et al. (1998) Anticancer Drugs, 9:105-123. The expression or activity of several different enzymes involved in repairing damaged DNA are altered in chemotherapeutic drug-resistant cells. For example, O6-alkylguanine-DNA alkyltransferase, a DNA repair enzyme, exhibits higher expression in cells less sensitive to alkylating chemotherapeutic drugs than in cell more sensitive to the drugs. Belanich et al. (1996) Cancer Res., 56:783-788. Similarly, DNA polymerase a, DNA polymerase xcex2, and topoisomerase II are elevated in some tumor cells that are resistant to chemotherapeutic drugs. Friedman et al. (1994) Cancer Res., 54:3487-93. In addition to these, many more DNA repair enzymes are aberrantly expressed in some drug-resistant tumor cells, including, for example: AP endonuclease and DNA glycosylases. See, Barrett et al., supra.
The present invention is based, at least in part, on the discovery of the human gene encoding DRT111. The apparent murine homolog of human DRT111 is expressed at a higher level in a cyclophosphamide-resistant variant of the murine tumor cell line EMT-6 CTX than in the EMT-6 cell line from which EMT-6 CTX tumor cells are derived. The human DRT111 cDNA described below (SEQ ID NO:1) has a 1203 nucleotide open reading frame (nucleotides 145-1348 of SEQ ID NO:1; SEQ ID NO:3) which encodes a 401 amino acid protein (SEQ ID NO:2). The cDNA encoding human DRT111 is 35% identical to the cDNA encoding Arabidopsis thaliana DRT111, a gene that encodes a protein thought to be involved in DNA damage repair. DRT111 nucleic acids and polypeptides, as well as molecules which increase or decrease expression or activity of DRT111, are expected to be useful in the diagnosis and treatment of disorders associated with aberrant DNA damage repair (e.g., drug-resistant cancer).
The DRT111 molecules of the present invention are useful as modulating agents in regulating a variety of cellular processes. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding DRT111 proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of DRT111-encoding nucleic acids.
The invention features a nucleic acid molecule which is at least 45% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to the nucleotide sequence shown in SEQ ID NO:1, or SEQ ID NO:3, or the nucleotide sequence of the cDNA insert of the plasmid deposited with ATCC as Accession Number (the xe2x80x9ccDNA of ATCC 209937xe2x80x9d), or a complement thereof.
The invention features a nucleic acid molecule which includes a fragment of at least 30 (50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1250, 1500, or 1695) nucleotides of the nucleotide sequence shown in SEQ ID NO:1, or SEQ ID NO:3, or the nucleotide sequence of the cDNA ATCC 209937, or a complement thereof.
The invention also features a nucleic acid molecule which includes a nucleotide sequence encoding a protein having an amino acid sequence that is at least 45% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to the amino acid sequence of SEQ ID NO:2, or the amino acid sequence encoded by the cDNA of ATCC 209937. In a preferred embodiment, DRT111 nucleic acid molecule has the nucleotide sequence shown SEQ ID NO:1, or SEQ ID NO:3, or the nucleotide sequence of the cDNA of ATCC 209937.
Also within the invention is a nucleic acid molecule which encodes a fragment of a polypeptide having the amino acid sequence of SEQ ID NO:2, the fragment including at least 15 (25, 30, 50, 100, 150, 300, or 400) contiguous amino acids of SEQ ID NO:2 or the polypeptide encoded by the cDNA of ATCC 209937.
The invention includes a nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide having the amino acid sequence of SEQ ID NO:2 or an amino acid sequence encoded by the cDNA of ATCC 209937, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3 under stringent conditions.
Also within the invention are isolated nucleic acid molecules which encode a polypeptide having the amino acid sequence of SEQ ID NO:2; isolated nucleic acid molecules which encode a fragment of a polypeptide having the amino acid sequence of SEQ ID NO:2, wherein the fragment contains at least 15 contiguous amino acids of SEQ ID NO:2; and isolated nucleic acid molecules which encode naturally occurring allelic variants of a polypeptide including the amino acid sequence of SEQ ID NO: 2, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, the complement of SEQ ID NO:1, or the complement of SEQ ID NO:3 under stringent conditions. These isolated nucleic acid molecules can have the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, the complement of SEQ ID NO:1, or the complement of SEQ ID NO:3; the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, the complement of SEQ ID NO:1 or, the complement of SEQ ID NO:3, wherein the xe2x80x9cTxe2x80x9ds are replaced with xe2x80x9cUxe2x80x9ds; or fragments of the foregoing that include at least 30 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:3, the complement of SEQ ID NO:1, or the complement of SEQ ID NO:3.
Another embodiment of the invention features isolated nucleic acid molecules which specifically detect DRT111 nucleic acid molecules relative to non-DRT111 nucleic acid molecules. For example, in one embodiment, such nucleic acid molecules hybridize under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or the cDNA of ATCC 209937, or a complement thereof. In another embodiment, these nucleic acid molecules are at least 30 (50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1250, 1500, or 1695) nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, the cDNA of ATCC 209937, or a complement thereof. In one embodiment, the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of a DRT111 nucleic acid.
Also included in the invention are isolated nucleic acid molecules having a nucleotide sequence which is at least 55% identical to the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3, or a complement thereof; those having a nucleotide sequence that hybridizes to a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3 under stringent conditions, or a complement thereof; and those having a nucleotide sequence that hybridizes to a nucleic acid molecule consisting of the nucleotide sequence of the cDNA insert of the plasmid deposited with ATCC as Accession Number 209937 under stringent conditions, or a complement thereof.
The nucleic acids of the invention can also include other nucleic acid sequences. In various embodiments, the nucleic acids of the invention further include vector nucleic acid sequences or nucleic acid sequences encoding a heterologogus polypeptide. Thus, the invention also includes a vector, e.g., a recombinant expression vector, which includes a nucleic acid molecule of the invention.
The invention also features host cells containing a nucleic acid molecule of the invention. In some cases, the host cell is a mammalian cell, such as a non-human mammalian cell. The invention also provides a method for producing a polypeptide of the invention (e.g., human DRT111 protein) by culturing, in a suitable medium, a host cell of the invention containing a recombinant expression vector such that a polypeptide of the invention is produced.
Another aspect of this invention features isolated or recombinant DRT111 proteins and polypeptides. Preferred DRT111 proteins and polypeptides possess at least one biological activity possessed by naturally occurring human DRT111, e.g., the ability to bind proteins involved in DNA repair, the ability to facilitate DNA repair, and the ability to impart cellular resistance to agents that induce DNA damage.
Polypeptides or proteins featured in the invention include: isolated polypeptides having an amino acid sequence that is at least about 45%, preferably 55%, 65%, 75%, 85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:2.
Also within the invention are: an isolated polypeptide which is encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 45%, preferably 55%, 65%, 75%, 85%, or 95% identical to SEQ ID NO:3 or the cDNA of ATCC 209937; and an isolated polypeptide which is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:3 or the non-coding strand of the cDNA of ATCC 209937.
Additional polypeptides of the invention include those encoded by isolated nucleic acid molecules which encode a polypeptide having the amino acid sequence of SEQ ID NO:2; isolated nucleic acid molecules which encode a fragment of a polypeptide having the amino acid sequence of SEQ ID NO:2, wherein the fragment contains at least 15 contiguous amino acids of SEQ ID NO:2; and isolated nucleic acid molecules which encode naturally occurring allelic variants of a polypeptide including the amino acid sequence of SEQ ID NO: 2, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, the complement of SEQ ID NO:1, or the complement of SEQ ID NO:3 under stringent conditions.
Other polypeptides of the invention include: a fragment of a polypeptide having the amino acid sequence of SEQ ID NO:2, wherein the fragment includes at least 17 contiguous amino acids of SEQ ID NO:2; a naturally occurring allelic variant of a polypeptide having the amino acid sequence of SEQ ID NO:2 or an amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC as Accession Number 209937, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3 under stringent conditions; and a polypeptide which is encoded by a nucleic acid molecule having a nucleotide sequence which is at least 57% identical to a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3.
Isolated polypeptides of the invention can have the amino acid sequence of SEQ ID NO:2 or an amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC as Accession Number 209937.
The polypeptides of the present invention, or biologically active portions thereof, can be operatively linked to heterologous amino acid sequences to form fusion proteins. The invention further features antibodies that specifically bind to the polypeptides of the invention, such as monoclonal or polyclonal antibodies. In addition, the polypeptides or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
Methods of producing polypeptides are also included within the invention. One such method includes the step of culturing a host cell containing an isolated nucleic acid molecule which encodes: a polypeptide having the amino acid sequence of SEQ ID NO:2; a fragment of a polypeptide having the amino acid sequence of SEQ ID NO:2, wherein the fragment includes at least 17 contiguous amino acids of SEQ ID NO:2; or a naturally occurring allelic variant of a polypeptide having the amino acid sequence of SEQ ID NO: 2, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, the complement of SEQ ID NO:1, or the complement of SEQ ID NO:3 under stringent conditions; under conditions in which the nucleic acid is expressed.
Another such method for producing polypeptides features the step of culturing a host cell having an isolated nucleic acid molecule encoding: a polypeptide having the amino acid sequence of SEQ ID NO:2 or an amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC as Accession Number 209937; a fragment of a polypeptide having the amino acid sequence of SEQ ID NO:2 or an amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC as Accession Number 209937, wherein the fragment includes at least 17 contiguous amino acids of SEQ ID NO:2 or an amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC as Accession Number 209937; or a naturally occurring allelic variant of a polypeptide having the amino acid sequence of SEQ ID NO:2 or an amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC as Accession Number 209937, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, a complement of SEQ ID NO:1, or a complement of SEQ ID NO:3 under stringent conditions; under conditions in which the nucleic acid molecule is expressed.
The invention also includes a method for detecting the presence of a polypeptide of the invention in a sample. This method features the steps of contacting the sample with a compound which selectively binds to the polypeptide and then determining whether the compound binds to a polypeptide in the sample. In some cases, the compound which binds to the polypeptide is an antibody.
Also within the invention is a kit including a compound which selectively binds to a polypeptide of the invention and instructions for use.
Additionally featured in the invention are methods for detecting the presence of a nucleic acid molecule of the invention in a sample. This method includes the steps of contacting the sample with a nucleic acid probe or primer which selectively hybridizes to a nucleic acid molecule of the invention; and then determining whether the nucleic acid probe or primer binds to a nucleic acid molecule in the sample. In many cases, the foregoing sample includes mRNA molecules or genomic DNA.
Also within the invention is a kit including a compound which selectively hybridizes to a nucleic acid molecule of the invention and instructions for use.
Other methods of the invention include those for identifying a compound which binds to a polypeptide featured in the invention. These methods include the steps of contacting a polypeptide of the invention with a test compound and then determining whether the polypeptide binds to the test compound. In various embodiments of these methods, the binding of the test compound to the polypeptide is detected using an assay which measures binding of the test compound to the polypeptide or using a competition binding assay.
The invention also includes a method for modulating the activity of a polypeptide of the invention. This method includes the steps of contacting the polypeptide or a cell expressing the polypeptide with a compound which binds to the polypeptide in a sufficient concentration to modulate the activity of the polypeptide.
In another aspect, the invention provides a method for identifying a compound that binds to or modulates the activity of a polypeptide of the invention (e.g., a DRT111 protein). In general, such methods entail measuring a biological activity of the polypeptide in the presence and absence of a test compound and identifying those compounds which alter the activity of the polypeptide. One such method includes the steps of contacting the polypeptide with a test compound and then determining the effect of the test compound on the activity of the polypeptide (i.e., upregulation or downregulation) to thereby identify a compound which modulates the activity of the polypeptide.
The invention also features methods for identifying a compound which modulates the expression of a nucleic acid or polypeptide of the invention by measuring the expression of the nucleic acid or polypeptide in the presence and absence of a compound.
Other aspects of the invention are methods and compositions relating to drug resistance. A xe2x80x9cdrug-resistant phenotypexe2x80x9d refers to a cellular phenotype which is associated with increased survival after exposure to a drug, e.g., a chemotherapeutic drug, compared to a cell that does not have this phenotype. A xe2x80x9cdrug-resistant cellxe2x80x9d refers to a cell that exhibits this phenotype.
Also within the invention is a method of determining whether a cell has a drug-resistant phenotype by measuring the expression of DRT111 in the cell and comparing this expression to that in a control cell. Increased expression of DRT111 in the cell compared to the control cell indicates that the cell has a drug-resistant phenotype. In one embodiment of this method, DRT111 expression is determined by measuring DRT111 protein. In another embodiment, DRT111 expression is measured using an antibody. In still another embodiment, DRT111 expression is measured by quantifying mRNA encoding DRT111 or the copy number of the DRT111 gene.
The invention also includes a method for modulating the drug resistance of a cell by modulating DRT111 expression or activity within the cell. Thus in one embodiment, the drug-resistance of a cell is reduced by contacting the cell with a molecule that reduces the expression of DRT111 within the cell (e.g., an antisense nucleic acid molecule)
Another aspect of the present invention is a method of improving effectiveness of chemotherapy for a mammal having a disorder associated with the presence of drug-resistant neoplastic cells. In this method, a chemotherapeutic drug and a molecule that reduces expression of DRT111 are co-administered to a mammal.
The invention also includes a method of identifying a compound that modulates the drug resistance of a cell by first contacting the cell with a test compound and then measuring and comparing DRT111 expression in the cell exposed to the compound to DRT111 expression in a control cell not exposed to the compound. The compound is identified as modulator of drug resistance when the level of DRT111 expression in the cell exposed to the compound differs from the level of DRT111 expression in cells not exposed to the compound. In one embodiment of this method, the cell has a drug-resistant phenotype. In another embodiment, the cell is a mammalian cell. This method may also include an optional step of measuring the drug resistance of the cell in the presence of the identified modulator of drug resistance. The DRT111 modulating compounds that are identified in the foregoing methods are also included within the invention.
The invention also features a method of treating a mammal suspected of having a disorder associated with the presence of drug-resistant cells. This method includes the steps of determining whether a mammal has a disorder associated with the presence of drug-resistant cells having increased DRT111 expression (e.g., drug-resistant cancer), and administering to the mammal a compound that sufficiently reduces the expression of DRT111 so that the drug resistance of the cells associated with the disorder is modulated.
Another feature of the invention is a method for treating a patient having a neoplastic disorder (e.g., cancer) by administering to the patient a therapeutically effective amount of a compound that decreases the expression of DRT111.
Also within the invention is a method for increasing drug resistance in a cell having an undesirably low level of DRT111 expression by administering a compound that increases the expression of DRT111.
Other features and advantages of the invention will be apparent from the following detailed description and claims.